Method for treatment of cataract with radical scavenger

ABSTRACT

The invention provides a method for treatment of cataract which comprises administering to a subject in need of such treatment, a radical scavenger in an amount effective in treatment of cataract selected from the group consisting of a reducing thiol derivative, or a disulfide derivative and a sulfide derivative thereof, and a pharmaceutical composition for treatment of cataract which comprises an anti-cataract agent containing a radical scavenger and fine particles, such as emulsions, nanocapsules, alubmin microspheres and liposomes, which carries an anti-cataract agent and has a lipophilic and positively charged phase on the surface thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for treatment of cataractwhich comprises administering a radical scavenger such as a reducingthiol derivative, or a disulfide derivative or a sulfide derivativethereof.

2. Description of the Prior Art

In recent years, the incidense rate of senile cataract has beenincreasing with the increase in the rate of aged people in thepopulation in Japan. Mechanisms of outbreak or progress of cataract havenot been elucidated; however, it is now thought that pathogenic mattercauses faculty in biological anti-oxidation system to generally growweak and then lens proteins aggultinate to generate opacity in the lens.The faculty in biological anti-oxidation system seems to grow weak bythe following mechanisms: (1) in the course of progress of cataract,generation of reaction system in which anti-oxidant in the lens isabnormally consumed; and (2) defluxion of glutatione (GSH) of reducingbiocomponents by diffusion with other components in the lens due toabnormal transpenetration of membrane of the lens. Opacity of the lensis also suggested to be caused by change of ion balance of Na/K andirreversibility of ion balance by Ca²⁺.

Aldose reductase inhibitors (ARIs) have been developed as anti-cataractagents for diabetic cataract and some effective compounds have beenproposed in animal experiments. However, one of potential ARIs killed 3patients in its clinical trial in the United States, therefore, thiskind of anti-cataract agent was stopped from being used. A possibleanti-cataract agent has been expected to be developed for a long time.

The inventors have studied as anti-cataract agents some inhibitors ofthe lowering of faculty in biological anti-oxidation system and thepresent invention was made in the course of that study.

SUMMARY OF THE INVENTION

The first object of the invention is to provide a radical scavengerselected from reducing thiol derivatives or disulfide derivatives andsulfide derivatives thereof as an anti-cataract agent.

Diethyldithiocarbamate (DDC) which is a reducing thiol derivative hasbeen confirmed as an anti-cataract agent by the inventors.

As DDC is disadvantageously hydrophilic, it can not penetrate into thecornea and is not transferred to aqueous humor and the crystalline lens.In addition, DDC is also easily oxidized and unstable. Itsbioavailability, therefore, is low and the anti-cataract activity of DDCcan not be sufficiently expressed.

Now, it has been found that disulfide derivatives and sulfidederivatives of the reducing thiol derivatives, which are chemicallystable and lipophilic and therefore can easily pass through the cornea,are useful as anti-cataract agents.

Thus, in the course of the reserch on some radical scavengers, whichhave high bioavailability and a stable reducing SH group, asanti-cataract agents, it has been found that Disulfiram (DSF,tetraethylthiuram disulfide, Antabuse) which has been used for manyyears in therapy for chronic alcoholism in the United States shows apotential anti-cataract effect. DSF is a dimer of DDC, of whichadministration methods and non-toxicity are already confirmed, andpossesses significant potential anti-cataract effect when compared withDDC.

Since (1) DSF is able to pass through the cornea due to itsfat-solubility and (2) it is known that one molecule of DSF produces twomolecules of DDC by catalytically reductive action of albumin and onemolecule from DDC binds to SH group of albumin and another molecule fromDDC is released, DSF which has easily passed through the cornea seems tobe converted to DDC by action of albumin in the cornea and the lens topresent an anti-cataract effect therein.

The second object of the invention is to provide a pharmaceuticalcomposition for treatment of cataract which comprises an anti-cataractagent and fine particles which enhance the bioavailability ofanti-cataract agent and the transportation to the target positions.

That is, DSF is a lipophilic substance and therefore, its ability ofpenetrate into the cornea is very high. On the other hand, it is notmiscible with water such as tear, and is very disadvantageously to beincorporated in an aqueous eye drop penetration. This problem was solvedby microcapsulation of DSF as fine particles such as emulsions,nanocapsules, albumin microspheres and liposomes.

The third object of the invention is to provide a positively chargedfine particle, which is used for transportation of anti-cataract agentinto the aqueous humor and the lens.

That is, since the surface of the cornea is negatively charged, fineparticles are treated with a positively charged substance to add apositive charge on the surface thereof. The fine particles thus treated,of which the charged surface is confirmed as positive by determinationof ζ potential, can easily bind to the negatively charged cornea,whereby anti-cataract agent in the fine particles can be positivelytransferred into the aqueous humor and the lens. Thus the inventors havedeveloped fine particles for anti-cataract agents with highbioavailability.

Therefore, the present invention provides; (1) a method for treatment ofcataract which comprises administering to a subject in need of suchtreatment, a radical scavenger in an amount effective in treatment ofcataract selected from the group consisting of a reducing thiolderivative, and a disulfide derivative or a sulfide derivative thereof,for example, compounds of formula (I) and (II), compounds of formula(III) or a cephem, and (2) a pharmaceutical composition for treatment ofcataract which comprises an anti-cataract agent containing a radicalscavenger, and fine particles, such as emulsions, nanocapsules, alubminmicrospheres and liposomes, which carries the anti-cataract agent andhas a lipophilic and positively charged phase on the surface thereof.

DETAILED DESCRIPTION OF THE INVENTION

The term "a reducing thiol derivative" includes compounds which areeasily oxidized, for example, compounds of formula: ##STR1## wherein R¹,R² and R³ are independently a straight or branched lower alkyl which maybe substituted by hydroxy, lower alkyloxy or lower alkyl carbonyloxy,their derivatives or a pharmaceutically acceptable salt thereof,preferably, diethyl dithiocarbamate, 1-methyl-1H-tetrazol-5-yl-thiol and1-(2-hydroxyethyl)-1H-tetrazol-5-yl-thiol or a pharmaceuticallyacceptable salt thereof.

Examples of "a disulfide derivative" which is derived from reducingthiols are compounds of formula: ##STR2## wherein R¹ and R² are asdefined above, derivatives thereof and pharmaceutically acceptable saltsthereof, preferably, DSF.

As examples of "a sulfide derivative" which is also derived from thereducing thiol derivatives, there can be mentioned cephems such ascefamandole, cefoperazone, cefmenoxime hemihydrochloride, cefmetazole,cefotetan, latamoxef, cefbuperazone, cefpiramide and flomoxef andpharmaceutically acceptable salts thereof which are the derivatives from1-methyl-1H-tetrazol-5-yl-thio and1-(2-hydroxyethyl)-1H-tetrazol-5-yl-thiol, preferably cefmetazole.

The term "Radical scavengers" which have high reactivities againstradicals, and can react with radicals rapidly, and can remove radicalsfrom the reaction system, and can terminate the subsequent reactions.More specifically, radical scavengers can trap the active oxygens suchas singlet oxygen, hydroperoxide, superoxide anion and hydroxyl radicaland suppress biological disorders by chain oxidation reactions relatedto these oxygens.

The term "treatment" includes the prevention of cataracts and cure ofthe disease.

The term "lower alkyl" in lower alkyloxy or lower alkyl carbonyloxygroup includes saturated and straight or branched hydrocarbon groupshaving 1 to 6, preferably 1 to 5, more preferably, 1 to 4 atoms. Forexample, there can be mentioned methyl, ethyl, propyl, isopropyl, butyland t-butyl.

Small particles such as liposomes, emulsions, nanocapsules and albuminmicrospheres are used as carriers for the anti-cataract agents.

Liposomes which are formed in water by dispersing natural lipids aresimilar to the structure of mammalian cell membranes and are used as amodel of artificial cell membrane. Therefore, there are many suggestionin medical and pharmaceutical fields that liposomes used as tissuetargeting drug carriers, artificial red blood cells, and cell-modifyingand enzyme-immobilizing substrates have good adaptations to livingbodies. However, while liposomes prepared by the ordinary methods havebeen applied to the purposes mentioned above, there were fewpreparations which could be used in practice. The reasons are for thatfirstly the liposomes prepared by the conventional method have lessstructural stability if they have good adaptation to tissues, and thatsecondly they did not have the for tissue and cell targeting which isvery important factor for drug carriers.

Therefore, to improve their weak points we modified liposomes withlipophilic positively charged substances, and the liposomes wereenhanced in their stability and targeting ability to objective tissuesand cells.

Though the techniques for the addition of surface charge to liposomeshave been available, the relationship between the added membrane chargeand drug transport of liposomes has not been clarified quantitatively.In this invention, the relationship between membrane charge modified bylipophilic positive substances and stability of liposomes was proved bydetermination of ζ-potential of liposomes and heat flow of liposomes bya differential scanning calorimetry. Then liposomes prepared by thismanner which show the controlled release of drugs and can transfer thedrugs to crystalline lens through cornea, can be used for treatment ofcataracts.

Accordingly, the liposomes to which are added positive charge by thetreatment of lipophilic positively charged substances and loadedanti-cataract agents, show cornea specific targeting and binding, goodtransability to transport drugs into cornea and, no toxicity against eyetissues. Therefore, it is suggested that the administration of thispreparation is an effective way to cure cataracts.

In the present invention, small particle carriers such as saidliposomes, emulsions, nanocapsules, and albumin microcapspheres whichare applied to pharmaceutical technology, can be used for cure againstcataracts.

Method for Preparation of Liposomes

A solution of vesicle-forming lipids in a solvent such as chloroform orether in a round-bottomed flask is evaporated under nitrogen to formthin film of lipid on the inside wall of the round-bottomed flask. Thedried film is hydrated by vortexing in water or buffers. Subsequentlythe resulting suspensions are sonicated using a probe for 1-3 min. Thesuspensions can be used as liposome preparations.

The preparation procedure of liposomes by the method of reverse-phaseevaporation follows. Several phospholipids, either pure or mixed withother lipids such as cholesterol, long-chain alcohols etc., can be usedwith similar results. The lipid mixture is added to a round-bottom flaskwith long extension neck. The aqueous phase is added, the system is keptcontinuously under nitrogen, and the resulting two-phase system issonicated briefly (a few minute) in a bath-type sonicator until themixture becomes either a clear one-phase dispersion or a homogeneousopalescent dispersion. The mixture is then placed on an rotaryevaporator and organic solvent is removed under reduced pressure. Theobtained dispersions can be used as liposome preparations.

In either of the two methods mentioned above, hydrophilic and lipophilicagents used as anti-cataract drugs are dissolved in appropriate buffersand in organic solvents, respectively.

Other methods besides the above two methods, such as French-pressmethod, freezed-dried method and freezed-thawed method can also be usedfor preparation of liposomes.

Liposomes presented in this patent can be prepared by conventionalmethods and those which have been reported already consist mainly ofphospholipids and cholesterol. Phospholipids such as egg york lecithin,soybean lecithin, phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,phosphatidic acid, and sphingomyelin etc., can be used for preparationof liposomes and liposomes can be prepared by using one or morecombinations of these lipids.

Liposomes of the present invention consist at least of said lipids andan anti-cataract agent mentioned in sections following, and then anappropriate amount of cholesterol may be added to said lipids dependingon the anti-cataract agent and lipophilic negatively or positivelycharged substances to be used, and cholesterol may be addedadvantageously when liposomes contain lipophilic negatively chargesubstances.

Method for Preparation of Emulsions

There are two types of emulsions i.e., water in oil (w/o) and oil inwater (o/w) types which are determined by emulsifier used. Emulsions areformed as small vesicles by mixing of oil and water. For example w/o/wemulsions are prepared by the method of two step emulsifying which in afirst step involves preparing w/o emulsions and in second step involveddispersing w/o emulsion it into water containing appropriate emulsifier.

Typical oil-in-water emulsions are prepared containing 10% (w/w) soybeenoil, various concentrations (0.6-2.0%(w/w)) of egg lecithins(phospholipids), and 2.5% (w/w) glycerol in a sufficient amount ofdistilled water. The emulsifiers (egg lecithins) are dissolved in theoil phase, heated to 80° C. in a tank, and water which had beenpreheated to 80° C. is added to the solution. The agitator used is anautohomomixer, a high shear mixer; the temperature of the mixture ismaintained at 80° C. for 30 min after the start of agitation in thetank. The impeller speed is kept at 10,000 rev/min. To make fineemulsions, coarse emulsions are introduced rapidly into a two-stagepressure homogenizer operating at 4500 psi. The obtained dispersions canbe used as emulsion preparations.

Method for Preparation of Microcapsules

Microcapsules are prepared by coating the surface of small drug vesiclesas core with polymers. Microcapsules are prepared by the method ofcoacervation and method of surface coagulation. In a coacervationmethod, core substances are dispersed in solution containing coatingmaterials (polymers), and the surface of core is coagulated withcoacervate, and the coagulate is hardened. Ethylcellulose, poly-lactate,polyvinylacetate, gelatin and starch etc. are used as coating materialsfor preparation of microcapsules.

Method for Preparation of Microspheres

The preparation of microcapsules mentioned above is one coatingtechnique. On the other hand, microspheres are dispersed as solution orcrystal of drugs in macromolecule matrix. Fundamentally microspheres areprepared by the method which comprises emulsifying of albumin solutionin cotton oil or organic solvent (w/o emulsion) and hardening such as byheating, chemical bridging, drying in water, or polymerization byirradiation. Biodegradable polymers such as albumin, gelatin, dextran,polylactate, polyethylenecarbonate, and non-biodegradable polymers suchas polystyrene, agarose, polyacrylamide etc. are used as matrix for thepreparation of microspheres. The particle sizes of microspheres can bechanged from 0.3 to 500 μm by using detergents.

Method for Preparation of Nanocapsules

Nanocapsules are small particles with nanometer order of diametercontaining drugs. In the same way of microcapsules, nanocapsules can beprepared by the methods as coacervation and micelle coagulation usinghigh molecular materials such as albumin, gelatin alkylcyanoacrylate.

Method for addition of Lipophilic Positive Charge into Small Particles

Addition of positive charge into small particles can be performed bymixing of lipophilic positively charged substances with membrane formingmatrix when preparing small particles simultaneously or thereafter.

The lipophilic positively charged substances of the invention includecetylpyridinium chloride (CPC), dimethyldialkyl (C₈ to C₁₈) ammoniumbromide (DC-1-8), N-methyl-N-(β-hydroxyethyl)-didodecyl ammonium bromide(DC-2-12), N-(α-trimethylammonioacetyl)-didodecyl-L-glutamate chloride(DC-3-12L), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride(DC-3-12L), N-(α-trimethylammonioacetyl)-O,O'-bis-(1H, 1H, 2H,2H-perfluorododecyl)-L-glutamate chloride (DC-5-8F2L), and as aminesalts of cationic detergents can be mentioned, for example, alkyl aminesalts, polyamine and alkanol amine fatty acid derivatives, alkylquartenary ammonium salts such as alkyl trimethyl ammonium salts,dialkyl trimethyl ammonium salts and alkyl dimethyl benzyl ammoniumsalts, cyclic quartenary ammonium salts such as alkyl pyridinium saltsand alkyl isoquinolinium salts, aromatic ammonium salts such asbenzethonium chloride, and nitrogen containing detergents including, forexample, polyoxyethylene fatty acid amide, polyoxyethylene alkyl amine,alkylol amide, and alkylamine oxide.

The lipophilic positively charged substance is added, for example, in anamount of 1 to 30% by mole, preferably 3 to 20% by mole, most preferably5 to 15% by mole based on the total fine particles components.

The lipophilic and positively charged fine particles containinganti-cataract agent of the present invention, which are prepared by themethod mentioned above, are used for preparing aqueous eye drops, aqeoussuspensions for eye drops, non-aqueous eye drops, non-aqeous suspensionsfor eye drops or eye ointments by conventional processes.

A unit dose depends on the severity of conditions of cataract and bodyweight or age of patients and like. Due to the low toxicity of theradical scavenger of the invention, the unit dose is not limited andpreferably is 1 μg to 1 mg and preferably a dose per day is 1 μg to 50mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show DDC release from liposomes containing DSF at pH8.0.

FIG. 2 shows an experimental device for cornea transpenetration.

FIG. 3. shows penetration of DDC releasing from DPPC/DMPC/CPC (2/8/1)liposomes containing DSF through a rabbit cornea.

FIG. 4 shows penetration of DDC releasing from DPPC/DMPC/CPC (2/8/1)liposomes containing DSF through rabbit cornea.

FIG. 5 shows effect of GE and DDC on GSH content in mouse crystallinelens treated with Diamide.

FIG. 6 shows changes of Na/K ratio in mice crystalline lenses treatedwith or without anti-cataract agents.

FIG. 7 shows changes of Ca contents in mice crystalline lenses treatedwith or without anti-cataract agents.

FIG. 8 shows inhibition activity of various drugs against aldosereductase from rat crystalline lens.

FIG. 9 shows scheimpflug and retroilumination images of X-irradiatedrats and effect of DSF against its cataract.

FIGS. 10A and 10B show anterior (FIG. 10A) and nuclear opacity (FIG.10B) in crystalline lens of X-irradiated rats treated with or withoutanti-cataract agents.

FIG. 11 shows posterior opacity in crystalline lenses of X-irradiatedrats treated with or without anti-cataract agents.

FIGS. 12A and 12B show effects of anti-cataract agents on Na/K ratio(FIGS. A) and Ca (FIG. B) contents in crystalline lenses of X-irradiatedrats treated with or without anti-cataract agents.

FIG. 13 shows effects of anti-cataract agents on GSH contents incrystalline lenses of X-irradiated rats treated with or withoutanti-cataract agents.

FIG. 14 shows photographs of selnite rats with or without instillationof DSF-liposomes, DSF-suspension and CMZ-liposomes.

FIG. 15 shows photographs of crystalline lenses of selnite rats with orwithout instillation of DSF-liposomes, DSF-suspension and CMZ-liposomes.

FIG. 16 shows scheimpflug and retroilumination images of selenitecataract rats.

FIGS. 17A and 17B show opacity (slit image, FIG. 17A) and transparency(retroillumination image, FIG. 17B) in crystalline lenses of seleniterats treated with or without anti-cataract agents.

FIGS. 18A and 18B show effects of anti-cataract agents on Na/K ratio(FIG. A) and Ca (FIG. B) contents in crystalline lenses of selenite ratstreated with or without anti-cataract agents.

FIG. 19 shows effects of anti-cataract agents on GSH contents incrystalline lenses of selenite rats treated with or withoutanti-cataract agents.

EXAMPLE Example 1 Method for Preparation of Liposomes Containing DSF

Liposomes containing DSF were prepared by the method of reverse phasesolvent evaporation with a minor modification. DSF 10 mg, DPPC, DMPC andCPC were dissolved in 20 ml of dichloromethane and this solution wasused as a hydrophobic phase. The hydrophobic phase was mixed with 10 mlof buffer solutions (A: Hind-Goyan's buffer, pH 6.5; B: Gifford'sbuffer, pH 8.0) and was sonicated under stream of nitrogen gas by usinga bath type sonicator (Branson Cleaning Equipment Company, U.S.A). Thew/o emulsion was aspirated for 30 min at 37° C. to remove CH₂ Cl₂. TheLiposomes were passed through an Excluder with membranes (pore sizes of1.0, 0.6, 0.2 and 0.1 μm), which repeated ten times for each membrane.The particle size of obtained liposomes was about 80 nm.

Preparation Method of Liposomes Containing Cefmetasol (CMZ)

Liposomes containing CMZ was prepared by the method mentioned aboveexcept of using CMZ dissolved in buffer solutions. DPPC, DMPC and CPCwere dissolved in 20 ml of dichloromethane and this solution was used asa lipophilic phase. The lipophilic phase was mixed with 10 ml of CMZsolution (1.0 g/ml) dissolved in buffer solutions (A: Hind-Goyan'sbuffer, pH 6.5; B: Gifford's buffer, pH 8.0) and was sonicated understream of nitrogen gas by using a bath type sonicator. The w/o emulsionwas aspirated for 30 min at 37° C. to remove CH₂ Cl₂. The Liposomes werepassed through an Excluder with membranes (pore sizes of 1.0, 0.6, 0.2and 0.1 μm), which repeated ten times for each membrane. To separate theliposomes from unencapsulated CMZ, the liposomes were chromatographed ona column Superose 6 (Pharmacia LKB Biotechnology AB, Sweden)equilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 145mM NaCl and 1 mM EDTA. The white turbid fractions were collected andused as a CMZ-liposome preparation. The particle size of obtainedliposomes was about 100 nm.

Determination of Encapsulation of DSF in Liposomes

The determination of DSF content loaded in the liposomes was carried outby following. Methanol was added to the liposomes obtained to disruptthe lipid membrane, and then it was mixed with an internal standardsolution and injected into HPLC apparatus. The liposomes were diluted by4 volumes of buffers such as Hind-Goyan's buffer (pH 6.5) and Giford'sbuffer (pH 8.0) and ultrafiltrated by using a Centrisert II. DSF infiltrate without liposomes was assayed by HPLC. However, no DSF in thesolution was measured and it was found that all of the drug added wasincorporated in liposome membranes.

Encapsulation Percentage of DSF-Liposomes

The encapsulation percentages of DSF in DSF-liposomes consisted ofvarious lipid compositions was shown in Table 1. Encapsulationpercentage of DSF in all liposomes shown in Table 1 were more than 90%.

                                      TABLE I                                     __________________________________________________________________________    Lipid Compositions and Encapsulation Percent of Liposomes                     Containing DSF                                                                             Content (mg/10 ml)                                                                         Encapsulation                                                                        ζ-Potential                             Liposomes    DPPC DMPC                                                                              CPC (%)    (mV)                                         __________________________________________________________________________    Dispersion in buffer (pH 6.5)                                                 DPPC/DMPC (5/5)                                                                            250  231 --  94.4   -13.47                                       DPPC/DMPC/CPC (5/5/1)                                                                      250  231 24.7                                                                              96.3   41.40                                        DPPC/DMPC/CPC (2/8/1)                                                                      90   336 22.1                                                                              100.7  48.12                                        Dispersion in buffer (pH 8.0)                                                 DPPC/DMPC/CPC (5/6/1)                                                                      250  231 24.7                                                                              92.0   --                                           DPPC/DMPC/CPC (2/8/1)                                                                      90   336 22.1                                                                              99.1   --                                           __________________________________________________________________________     DPPC: dipalmitoyl phosphatidylcholine, DMPC: dimyristoyl                      phosphatidylcholine,                                                          CPC: cetylpyridinum chloride.                                            

ζ-Potential of Liposomes Containing DSF

ζ-Potential of liposomes containing DSF was determined by using a LazarZee Meter Model 501 (Pen Kem Comp., Ltd., USA). The liposomes werediluted by 150 volumes of 5 mM potassium phosphate buffer (pH 6.5) andits ζ-potential was determined at 150 V. The ζ-potential was correctedfor change of temperature by following a equation.

    ζ-potential (corrected)=ζ-potential (determined)×(1-0.02T)

CPC in liposomes was added for enhancement of positive charge on surfaceof liposome membrane. The addition of one molar ratio of CPC againstlipids rose to 40-50 mV from about -13 mV (liposomes without CPC).

Determination of DDC Release from Liposomes Containing DSF

The reaction mixture consisted of 0.24 ml of liposomes containing DSFdiluted 2.5-fold with Gifford's buffer (pH 9.5, 8.0 and 6.5) and 0.25 mlof bovine serum albumin (BSA) solution (1-7%) in same buffers. Thereaction was performed at 35° C. for 0-6 hr and terminated by cooling inthe ice bath for 3 min. The reaction mixture was ultrafiltrated by usingCentrisart II (Sartorius AG, Germany) and the filtrate was used as asample for HPLC.

DDC Release from Liposomes Containing DSF

(1) Release in Gifford buffer (pH 8.0)

FIGS. 1A and 1B show DDC release profiles from the liposomes in 3different concentrations of BSA solutions, 1.0, 1.75, and 3.5%, at pH8.0 by using Centrisart II which includes membrane to separate the DDCreleased from the liposomes in the incubation mixture. DPPC/DMPC/CPC(5/5/1, molar ratio) liposomes had a higher CDDC release rate thanDPPC/DMPC/CPC (2/8/1) liposomes (FIG. 1).

(2) Release in Hind-Goyan's buffer (pH 6.5)

No DDC release from all liposomes tested was observed at pH 6.5.

Experiment of In Vitro Transcorneal Penetration

Male rabbit, 2.0 kg of body weight was sacrificed by a injection oflethal dose of pentobarbital into a car vein and then two eyeballs wereremoved from cash rabbit. The cornea was set to the cell in which donorside was filled with liposomes containing DSF and in reservoir side with10 mM HEPES buffer (pH 7.4) containing 136 mM NaCl, 5.3 mM KCl, 1.0 mMK₂ HPO₄, 1.7 mM CaCl₂ and 5.5 mM glucose, shown in FIG. 2. Thepenetration was carried out at 37° C. for 0-6 hr. The sample (50 μl) waswithdrawn from reservoir side at the indicated times. DDC in the samplewas determined by the HPLC method (FIG. 2).

In Vitro Transcorneal Penetration of DDC from Liposomes Through RabbitCornea

(1) Penetration at pH 8.0

FIG. 3 shows DDC penetration profiles through rabbit corneas at pH 8.0About one-half hour after incubation, DDC from the liposomes wasobserved in the reservoir solution. Addition of 1.75% BSA in donorsolution did affect on the DDC penetration (FIG. 3).

(2) Penetration at pH 6.5

FIG. 4 shows the DDC penetration profiles at pH 6.5. Six hours afterincubation, DDC content in the reservoir solution was one third of thatat pH 8.0 (FIG. 4).

Example 2 Procedure Using Microspheres Containing DSF Using Copoly(Lactic/Glycolic) acid (LGA)

LGA-7515 (Wako Pure Chemical Industries, Ltd.) microspheres containingDSF were prepared by an in-water drying method. One hundred milligram ofDSF, 850 mg of LGA-7515 and 50 mg of CPC were dissolved in 12 ml ofmethylene chloride. The solution was then poured into 150 ml of 0.5%(w/v) polyvinyl alcohol solution under stirring at 800 rpm by means ofmagnetic stirrer. The stirring continued for 2 hr at 25±2° C. toevaporate off methylene chloride. The microspheres were passed throughstainless steel sieves (mesh size: 297, 177, and 75 μm). Themicrospheres were collected by filtration through a stirred glass disk,washed with purified water, and dried under reduced pressure at roomtemperature. The encapsulation percent of DSF in the microspheres wasabout 90% and its diameter was 1.0±0.3 μm.

Example 3 Procedure of Albumin Microspheres Containing DSF

Fine power (250 mg) of DSF was suspended in solution of bovine serumalbumin (250 mg/ml of purified water). The suspension was added into 100ml of cotton oil containing 10 (v/v) % Span 85, and stirred for 10 minat 250 rpm by glass stirrer, and emulsified by a Sonicator, ShimadzuUSP-600 (100 w, 30 min). This micro-emulsion (w/o type) was added into abath containing glycerine at 100° C. and then microsphere was formed.Subsequently this microsphere was washed of glycerine by organicsolvents such as ether etc. Encapsulation percent of DSF in themicrosphere obtained was about 95%. Average particle size of thismicrosphere was 1±0.5 μm.

Example 4 Pharmaceutical Preparations

The liposomes containing DSF prepared by the method of Experiment 1 orthose diluted with solutions containing preservatives and tonicityagents were used as eye drop preparations.

Determination of In Vitro Anti-Cataract Activities of Various Agents

(1) Method

Male ddY mice, 6 weeks old, were killed by decapitation, and the globeswere excised. The lenses were removed by a posterior approach and thentransferred on a nylon net to an petri containing D-MEM medium (lowglucose). The lenses were incubated at 37° C. for 2 hr and cleartransparent lenses were collected to be used for following experiments.The lenses were incubated at 37° C. for 1.5 hr in 3 ml of isotonic HEPESbuffer (10 mM hepes, 139 mM NaCl, 5.4 mM KCl, 1 mM K₂ HPO₄, 1.7 mM CaCl₂--H₂ O, pH 7.4) containing 1 mM diamide. Those were transferred to anincubation test tube containing 3 ml of isotonic HEPES buffer (pH 7.4)containing 5.5 mM glucose with 1 mM DDC or GE (glutathione isopropylester). The diamide treated and non-treated controls were treated by the5 hr-incubation without the above agents. The wet weight of thesetreated lenses were determined.

(2) Determination of GSH in Mice Lenses

The treated lenses were homogenized with 0.5 ml of purified water byTeflon homogenizer. GSH in the homogenate was determined by the methodof Beutler using DTNB and the HPLC method followed.

Fifty microliter of the lens homogenate were added into 100 μl ofacetonitrile containing 100 μg of sodium pantothenate (internalstandard) and centrifuged at 12,000 rpm for 10 min. The supernatantsolution (10 μl) was injected into a HPLC apparatus, Shimadzu LC-10ADequipped with a column oven, CTO-6A and the chromatography wereperformed under the conditions: a column of Supersphere 100RP-18(particle size: 4 μm, column size: 4.0×250 mm), mobile phase of 5%methanol containing 1.0% TFA, flow rate of 1 ml/min, and columntemperature at 35° C.

(3) Effect of DDC and GE on GSH Content in Diamide Treated Lens

GSH contents in the treated lenses determined by DTNB method were shownin Table 2 and FIG. 5.

                  TABLE 2                                                         ______________________________________                                        Effect of Anti-Cataract Agents on GSH Contents in Diamide-                    Treatment Mice Lenses (DTNB Method)                                                          GSH content (μg/lens)                                                                    Deviation                                        ______________________________________                                        Contol         10.3          0.5                                              Diamide treatment                                                             None           7.6           0.3                                              1 mM GE        10.3          0.8                                              1 mM DDC-NA    9.8           0.8                                              ______________________________________                                    

The GSH contents were 10.3±0.5 μg/lens in non-treated lenses and 7.3μg/lens in diamide-treated lenses. Diamide caused a rapid oxidation oflens GSH. By the diamide treatment there was a 30 percent decrease intotal GSH. On the other hand, total GSH in lenses treated with 1 mM DDCand GE were restored by the control GSH level (FIG. 5).

GSH contents in the treated lenses determined by HPLC method are shownin Table 3 and FIG. 5.

                  TABLE 3                                                         ______________________________________                                        Effect of Anti-Cataract Agents on GSH Contents in Diamide-                    Treatment Mice Lenses (HPLC Method)                                                          GSH content (μg/lens)                                                                    Deviation                                        ______________________________________                                        Control        4.4           0.3                                              Diamide treatment                                                             None           3.1           0.2                                              1 mM GE        4.0           0.2                                              1 mM DDC-NA    3.9           0.4                                              ______________________________________                                    

All of data were about half of those obtained by DTNB method. However,restoring of GSH by the treatment of DDC and GE were observed by thismethod also (FIG. 5)

Effect of DDC and GE on Na⁺ /K⁺ Ratio and Ca²⁺ Contents inDiamide-Treated Mice Lenses

(1) Methods

Male ddY mice, 6 weeks old, were killed by decapitation, and the gloveswere excised. The lenses were removed by a posterior approach and thentransferred on a nylon net to a petri dish containing D-MEM medium (lowglucose). The lenses were incubated at 37° C. for 19-20 hr in CO₂incubator under 5% CO₂ -air and clear transparent lenses were collectedto be used for following experiments. The lenses were incubated at 37°C. for 1.5 hr in 3 ml of isotonic HEPES buffer (10 mM HEPES, 136.2 mMNaCl, 5.3 mM KCl, 1.0 mM K₂ HPO₄, 1.7 mM CaCl₂ ·2H₂ O, 5.5 mM glucose pH7.4) containing 1 mM diamide and the HEPES buffer without glucose. Thosewere transferred to an incubation test tube containing 3 ml of isotonicHEPES buffer (pH 7.4) with 1 mM DDC or GE. The diamide treated andnon-treated controls were treated by the 5 hr-incubation without theabove agents. The wet weight of these treated lenses were determined.Lenses were dried in vacuo at 100° C. for 8 hr, and then digested in 60%nitric acid (100 μl/lens) at 80° C. to degrade organic substances. Onemilliliter of purified water was added to the digests, and centrifugedat 300 xg for 10 min. Two tenth ml of the supernatant was mixed with 2ml of purified water, and was used for measurement of Na³⁰ and K⁺. Onetenth milliliter of 100 μg/ml lanthanum chloride was added to theresidual supernatant and used for measurement of Ca²⁺, Na⁺, K⁺ and Ca²⁺contents in the lenses were determined by atomic absorption spectrometer(Hitachi 180-80, Tokyo, Japan).

Effects of DDC and GE on Na⁺ /K⁺ Ratio and Ca²⁺ Contents inDiamide-Treated Mice Lenses

                  TABLE 4                                                         ______________________________________                                                 Contents (mmol/kg of H.sub.2 O)                                               Na.sup.+  K.sup.+   Ca.sup.2+                                                                             Na/K ratio                               ______________________________________                                        Diamide (-)                                                                            22.03 ± 3.39                                                                         95.90 ± 3.49                                                                         1.20 ± 0.27                                                                        0.23 ± 0.04                           Diamide (+)                                                                             60.38 ± 13.49                                                                        61.29 ± 13.78                                                                       1.98 ± 0.27                                                                        1.11 ± 0.04                           DDC-Na   36.56 ± 4.38                                                                         86.25 ± 4.02                                                                         1.09 ± 0.33                                                                        0.43 ± 0.07                           GE       51.60 ± 9.57                                                                         79.67 ± 8.63                                                                         1.39 ± 0.30                                                                        0.67 ± 0.20                           CMZ       40.59 ± 10.16                                                                        84.91 ± 10.06                                                                       1.11 ± 0.36                                                                        0.50 ± 0.19                           ______________________________________                                    

Effects of various agents on Na³⁰ /K⁺ ratio and Ca²⁺ contents indiamide-treated mice lenses are shown in Table 4 and FIG. 6. Thetreatments of 1 Mm DDC, GE, DTT and CMZ restored completely the Na⁺ /K⁺ratio and Ca²⁺ content to the control levels (FIG. 6).

Inhibitory Activities of Various Agents Against Rat Lens AldoseReductase

(1) Methods

Aldose reductase was prepared from rat lenses in the following manner.Lenses (20 to 30) were homogenized in 20 volumes of 5 mM sodiumphosphate buffer (pH 7.4), followed by centrifugation at 18,000 xg for 5min to remove insoluble materials. Solid ammonium sulfate was added tothe supernatant fluid to 30% saturation. The suspension was centrifugedat 18,000 xg for 10 min and the supernatant was recovered. Aldosereductase was precipitated from 30% saturated solution by the additionof solid ammonium sulfate to 75% saturation, and obtained bycentrifugation. The precipitate was collected and suspended in 3.2Mammonium sulfate.

The enzyme solution was assayed by following the absorbance at 340 nm ona Shimadzu UV-2000 spectrophotometer equipped with atemperature-controlled cuvette chamber. The solution (0.1 ml) was addedto a cuvette containing phosphate buffer (1.6 ml, pH 6.2, 0.1M finalconcentration) and NADPH (0.1 ml, 0.25 mM final concentration), and thecuvette was inserted into the spectrophotometer. The reaction wasstarted by the addition of DL-glyceraldehyde (0.1 ml, 50 mM finalconcentration) to the cuvette, and the decrease of absorbance at 340 nmfor 5 min at 25° C. was measured. The reaction was linear for at least 8min. Agents to be tested were dissolved with a minimal amount ofdimethylsulfoxide and diluted to the desired concentration with purifiedwater. The resulting solution (0.1 ml) was added to the cuvette. Thereference blank (to correct for non-specific oxidation of NADPH andabsorption of the agents) was prepared by using water instead ofDL-glyceraldehyde solution.

(2) Results

Inhibitory activities of various agents against rat lens aldosereductase are shown in Table 5 and FIG. 8. Quersitrine, pirenoxine, DSF,CMZ and GE showed lower inhibitory activities and trace or no inhibitoryactivity was observed in other agents (Table 5 and FIG. 8).

                  TABLE V                                                         ______________________________________                                        Inhibitory Activities (IC.sub.50) of Anti-Cataract Agents against             Aldose Reductase from Rat Crystalline Lens                                    Agent            Range of concentration                                                                       IC.sub.50 (μM)                             ______________________________________                                        Quercitrin       0.1˜1.0 μM                                                                          0.26                                          Pirenoxine (Catalin)                                                                           2.5˜25 μM                                                                           17.5                                          Disulfirum (DSF) 10˜200 μM                                                                           120                                           Cefmetazon (CMZ) 0.05˜1.00 mM                                                                           450                                           Glutathione isopropyl ester (GE)                                                               0.65˜0.85 mM                                                                           760                                           Frusultiamine (Alinamine F)                                                                    0.13˜1.25 mM                                                                           1.00 × 10.sup.3                         Tiopronin        0.50˜2.00 mM                                                                           1.35 × 10.sup.3                         Captopril        0.50˜2.00 mM                                                                           1.45 × 10.sup.3                         Glutathione (GSH)                                                                              0.5˜2.00 mM                                                                            1.55 × 10.sup.3                         Sodium diethyldithiocarbamate                                                                  0.5˜2.00 mM                                                                            2.60 × 10.sup.3                         (DDC--Na)                                                                     Glutathione disulfide (GSSG)                                                                   0.5˜2.00 mM                                                                            3.00 × 10.sup.3                         Thiamine hydrochloride                                                                         0.13˜0.63 mM                                                                           N.D.                                          S-Methyl glutathione                                                                           1.00 mM        N.D.                                          S-Ethyl glutathione                                                                            1.00 mM        N.D.                                          S-Buthyl glutathione                                                                           0.05 mM        N.D.                                          S-(n-Propyl) glutathione                                                                       0.05 mM        N.D.                                          ______________________________________                                    

In Vivo Anti-Cataract Experiment of DSF-Liposomes Using X-IrradiatedRats

In this in vivo anti-cataract experiment, the liposomes prepared by themethod of Experiment 1 was used. It was well known that the course ofdevelopment of X-ray induced cataracts are similar to that of humansenile cataract.

(1) Materials and Method

A. X-irradiation to Rats

The lenses of male Brown Norway Rats, 9 weeks old, was chosen as theexperimental model. Both eyes of each animal were irradiated at 10 Gy byusing a dose of 200 kVp X-rats, filtered with 0.5 mm Cu and 0.5 mm Al(HVL=1 mm of Cu). The animal bodies except the head was sealed with alead sheet (1 mm of thickness) for avoiding its systemic disorder byX-ray.

B. Instillation of Drugs

Each 10 μl of liposomes containing DSF (1 mg/ml), DDC solution (1mg/ml), GE solution (2 mg/ml) and CMZ solution (2.5 mg/ml) was instilled3 times per day to both eyes of the rats which were one month afterX-irradiation. The instillation was carried out for 6 months and theimage analysis for anterior eye of rats was performed at monthlyintervals by Nidek EAS-1000 Sheimpflug camera.

C. Image Analysis

Opacities of lenses of X-ray cataracts were digitized from the data ofdensitometry integrated area of the Scheimpflug images and were dividedinto 3 parts which were anterior, nuclear and posterior potions.Transparencies of the lenses were digitized from the data of theretroillumination images.

D. Determination of GSH in Rat Lenses

The treated lenses were homogenized with 0.5 ml of purified water byTeflon homogenizer. GSH in the homogenate was determined by the HPLCmethod followed.

Fifty microliter of the lens homogenate were added into 100 μl ofacetonitrile 100 μg of sodium pantothenate (internal standard) andcentrifuged at 12,000 rpm for 10 min. The supernatant solution (10 μl)was injected into a HPLC apparatus, Shimadzu LC-10AD equipped with acolumn oven, CTO-6A and the chromatography were performed under theconditions: A column of Supersphere 100RP-18 (particle size: 4 μm,column size: 4.0×250 mm), mobile phase of 5% methanol containing 1.0%TFA, flow rate of 1 ml/min, and column temperature at 35° C.

E. Determination of Na⁺, K⁺ and Ca²⁺ in Rat Lenses

Lenses were dried in vacuo at 100° C. for 8 hr, and then digested in 60%nitric acid (100 μl/lens) at 80° C. to degrade organic substances. Onemilliliter of purified water was added to the digests, and centrifugedat 3000 xg for 10 min. Two tenth ml of the supernatant was mixed with 2ml of purified water, and was used for measurement of Na⁺ and K⁺. Onetenth milliliter of 100 μg/ml lanthanum chloride was added to theresidual supernatant and used for measurement of Ca²⁺, Na⁺, K⁺ and Ca²⁺contents in the lenses were determined by atomic absorption spectrometer(Hitachi 180-80, Tokyo, Japan).

(2) Results

Evaluation of Anti-Cataract Effect of Instillation of DSF-Liposomes onX-Ray Cataracts by Image Analysis

FIG. 9 shows the images obtained from rat eyes 6 months afterX-irradiation by a Scheimpflug camera. The lenses of X-irradiationcontrol rats were observed strongly opaque in anterior and posteriorportions. However, the lenses of DSF-liposome instilled rats wereobserved to have no or a trace opacity at the two portions mentionedabove (FIGS. 9, 10A,B, 11).

Table 6 show the digitized data from the above results analyzed by aMackintosh program software, NIH Image. The opacities in anterior andposterior portions of lenses with the instillation of DSF-liposomes werelower than those of lenses without drug treatment, though thisdifference was not significant. Its reason is thought to be that therewere high individual differences of responses against X-ray among ratsused in this experiment, or the fixings of rat bodies may be not enoughto uniformly receive X-irradiation.

                  TABLE 6                                                         ______________________________________                                        Anterior, Nuclear and Posterior Opacities in Lenses of X-                     irradiated Pats Treated with or without Anti-Cataract Agents                           Opacity (count)                                                               Anterior portion                                                                          Nuclear portion                                                                           Posterior portion                            ______________________________________                                        Norm. Control                                                                          1206 ± 759                                                                             203397 ± 37889                                                                         1818 ± 1728                               X-Irradiation                                                                 None     4630 ± 1736                                                                            259396 ± 25752                                                                         12720 ± 9449                              DSF-liposomes                                                                          3515 ± 1656                                                                            279951 ± 32227                                                                         5716 ± 3798                               DDC-Na   4707 ± 1494                                                                            295814 ± 42134                                                                         9347 ± 5658                               solution                                                                      GE solution                                                                            3897 ± 2299                                                                            249074 ± 55147                                                                         7787 ± 6704                               CMZ solution                                                                           4578 ± 2214                                                                            264612 ± 52206                                                                         12304 ± 10843                             ______________________________________                                    

Effects of Instillation of DSF-Liposomes on Na⁺ /K⁺ Ratio and Ca²⁺Content in Crystalline Lenses of X-irradiated Rats

Effects of DSF-liposomes on Na⁺ /K⁺ ratio and Ca²⁺ contents anX-irradiated rat lenses are shown in Table 7 and FIG. 12. The treatmentsof DSF-liposomes restored completely the Na⁺ /K⁺ ratio and Ca²⁺ contentsto the control levels (FIGS. 12A,B)

                  TABLE 7                                                         ______________________________________                                        Na.sup.+ /K.sup.+  ratios, Ca.sup.2+  and GSH Contents in Lenses of           X-irradiated                                                                  Rats Treated with or without Anti-Cataract Agents                                                  Ca.sup.2+  content                                                                        GSH content                                           Na.sup.+ /K.sup.+ ratio                                                                   (mmol/kg H.sub.2 O)                                                                       (μmol/lens)                               ______________________________________                                        Control  0.26 ± 0.06                                                                            1.2 ± 0  135.2 ± 12.0                              X-Irradiation                                                                 None     0.43 ± 0.11                                                                            4.2 ± 2.3                                                                              100.1 ± 12.0                              DSF-liposomes                                                                          0.35 ± 0.08                                                                            4.4 ± 1.6                                                                              137.3 ± 22.6                              DDC-NA   0.37 ± 0.15                                                                            4.2 ± 1.3                                                                              126.7 ± 25.3                              solution                                                                      GE solution                                                                            0.30 ± 0.06                                                                            2.7 ± 1.3                                                                              130.6 ± 29.9                              CMZ solution                                                                           0.47 ± 0.20                                                                            4.0 ± 2.3                                                                               99.7 ± 21.7                              ______________________________________                                    

Effect of Instillation of DSF-Liposomes on GSH Content in Lenses ofX-irradiated Rates

GSH contents in the X-irradiated lenses determined by HPLC method areshown in Table 3 and FIG. 13. Restoring of GSH by the instillation ofDSF-liposome was observed significantly (FIG. 13).

In Vivo Anti-cataract Experiment of Liposomes Containing DSF UsingSelenite Cataract Rats

In this experiment, selenite-induced cataract rats were used as a modelsof acute cataracts, which relate disorders by radical derivativesgenerated during various metabolism in mammalian organs.

(1) Materials and Methods

A. Induction of Cataract by Inject of Selenite

Wistar rat pups post natal age 16-18 days were used in this experiment.The injection dose of sodium selenite was 3.28 mg/kg (19 μmol/kg).

B. Image Analysis

Lens opacification was observed by a Scheimpflug camera and recorded atselected time points from 0 to 7 days.

Image Analysis

Opacities of lenses of selenite cataracts were digitized from the dataof densitometry integrated areas of the Scheimpflug images.Transparencies of the lenses were digitized from the data of theretroillumination images.

C. Determination of GSH in Rat Lenses

The excised lenses were homogenized with 0.5 ml of purified water byTeflon homogenizer. GSH in the homogenate was determined by the HPLCmethod followed. Fifty microliter of the lens homogenate were added into100 μl of acetonitrile containing 100 μg of sodium pantothenate(internal standard) and centrifuged at 12,000 rpm for 10 min. Thesupernatant solution (10 μl) was injected into a HPLC apparatus,Shimadzu LC-10AD equipped with a column oven, CTO-6A and thechromatography were performed under the conditions; a column ofSupersphere 100RP-18 (particle size: 4 μn, column size: 4.0×250 mm),mobile phase of 5% methanol containing 1.0% TFA, flow rate of 1 ml/min,and column temperature at 35° C.

D. Determination of Na⁺, K⁺ and Ca²⁺ in Rat Lenses

Lenses were dried in vacuo at 100° C. for 8 hr, and then digested in 60%nitric acid (100 μl/lens) at 80° C. to degrade organic substances. Onemilliliter of purified water was added to the digests, and centrifugedat 300 xg for 10 min. Two tenth ml of the supernatant was mixed with 2ml of purified water, and was used for measurement of Na⁺ and K⁺. Onetenth milliliter of 100 μg/ml lanthanum chloride was added to theresidual supernatant and used for measurement of Ca²⁺, Na⁺, K⁺ and Ca²⁺contents in the lenses were determined by atomic absorption spectrometer(Hitachi 180-80, Tokyo, Japan).

(2) Results

Photographs for Anti-Cataract Effect of Instillation of Drugs onSelenite Cataracts

FIGS. 14 and 15 show photographs for heads and excised lenses ofselenite-induced rats, respectively. High level nuclear cataract wereobserved in the eyes of selenite treated rat, but no opacity wasobserved in those instilled eye drops containing DSF-liposomes. Both DSFsuspension and CMZ-liposome instilled eyes showed low level nuclearcataract (FIGS. 14 and 16).

Evaluation of Anti-Cataract Effect of Instillation of Drugs on SeleniteCataracts by Image Analysis

FIG. 16 shows the images obtained from rat eyes a week after injectionof selenite by a Scheimpflug camera. The lenses of selenite-treated ratsshowed strongly opacity in the nuclear portion. On the other hand, thelenses of DSF-liposome instilled rats showed no or a trace opacity atthe nuclear portion. The transparency of selenite-treated rat eyesdecreased by 60% of those of normal control rats. One the other hand,the transparency of DSF-instilled rat eyes was similar to control rateyes.

Table 8 show the digitized data from the above results analyzed by aMacintosh software, NIH Image. The opacities in the nuclear portion oflenses with instillation of DSF-liposomes were significantly lower thanthose of lenses without drug treatment (FIGS. 16 and 17A and 17B)

                  TABLE 8                                                         ______________________________________                                        Opacities and Transparencies in Lenses of Selenite-induced                    Cataract Rats Treated with or without Anti-Cataract Agents                                   Opacity     Transparency                                                      (count)     (%)                                                ______________________________________                                        Normal Control  3301 ± 1021                                                                           95.60 ± 4.01                                    Selenite Treatment                                                            None           20683 ± 4659                                                                           56.24 ± 17.08                                   DSF-Liposomes  10170 ± 6305                                                                           87.41 ± 9.72                                    DSF-Suspension 17017 ± 7195                                                                           69.74 ± 15.26                                   CMZ-Liposomes  19617 ± 6163                                                                           70.12 ± 12.64                                   ______________________________________                                    

Effects of Instillation of DSF-Liposomes on Na⁺ /K⁺ Ratio and Ca²⁺Content in Lenses of Selenite Cataract Rats

Effects of DSF-liposomes on Na⁺ /K⁺ ratio and Ca²⁺ contents in selenitecataract lenses are shown in Table 9 and FIG. 18. The instillation ofDSF-liposomes restored completely the Ca²⁺ content to the normal levels.However, no differences were observed between the Na⁺ /K⁺ ratios inlenses with and without anti-cataract agents and the ratios were almostsame as that in normal rat lens (FIG. 18A and B)

                  TABLE 9                                                         ______________________________________                                        Na.sup.+ /K.sup.+  ratios, Ca.sup.2+  and GSH contents in Lenses of           Selenite-                                                                     Induced Rats Treated with or without Anti-Cataract Agents                                          Ca.sup.2+  content                                                                        GSH content                                            Na.sup.+ /K.sup.+  ratio                                                                 (mmol/kg H.sub.2 O)                                                                       (μmol/lens)                               ______________________________________                                        Normal Control                                                                         0.11 ± 0.01                                                                            0.3 ± 0.1                                                                              125.1 ± 10.1                              Selenite                                                                      Treatment                                                                     None     0.16 ± 0.05                                                                            1.0 ± 0.5                                                                              99.0 ± 5.4                                DSF-Liposomes                                                                          0.13 ± 0.03                                                                            0.5 ± 0.2                                                                              111.0 ± 11.3                              DSF-Suspension                                                                         0.16 ± 0.02                                                                            0.5 ± 0.1                                                                              113.0 ± 14.2                              CMZ-Liposomes                                                                          0.14 ± 0.02                                                                            0.5 ± 0.1                                                                              110.7 ± 8.3                               ______________________________________                                    

Effects of Instillation of DSF-Liposomes on GSH Content in Lenses ofSelenite Cataract Rats

GSH contents in the selenite cataract lenses determined by the HPLCmethod were shown in Table 8 and FIG. 19. Restoring of GSH by theinstillation of DSF-liposomes, DSF suspension and CMZ-liposomes wasobserved significantly.

What is claimed is:
 1. A pharmaceutical composition for treatment ofcataract which comprises fine particles selected from the groupconsisting of emulsions, nanocapsules, albumin microspheres andliposomes, carrying an anti-cataract agent containing a radicalscavenger comprising a disulfide derivative of formula: ##STR3## whereinR¹ and R² are independently a straight or branched lower alkyl which maybe substituted by hydroxy, lower alkyloxy or lower alkyl carbonyloxy. 2.The pharmaceutical composition of claim 1 in which said disulfidederivative is disulfiram.
 3. The pharmaceutical composition according toclaim 1 in which the surface of said fine particles has a lipophilic andpositively charged phase.
 4. The pharmaceutical composition according toclaim 1 in which said fine particles include a lipophilic and positivelycharged substance in an amount of about 1 to 30% by mole of said fineparticles.
 5. The pharmaceutical composition according to claim 1 inwhich said fine particles are liposomes.
 6. The pharmaceuticalcomposition according to claim 5 in which said liposomes include naturalor semisynthetic phospholipid.
 7. The pharmaceutical compositionaccording to claim 1 in which said fine particles are produced bydissolving a fine particle forming material and a lipophilic andpositively charged substance in an amount of about 1 to 30% by mole ofthe total component in an organic medium, and then, in case of saidanti-cataract agent being lipophilic, further dissolving saidanti-cataract agent in the resultant organic medium, and in case of saidanti-cataract agent being water-soluble, dissolving said anti-cataractagent in an aqueous medium and adding the resultant aqueous medium tothe resultant organic medium, and in each case forming fine particles toobtain fine particles of which the surface has a lipophilic andpositively charged phase.
 8. A pharmaceutical composition for treatmentof cataract which comprises fine particles selected from the groupconsisting of emulsions, nanocapsules, albumin microspheres andliposomes, carrying an anti-cataract agent containing a radicalscavenger comprising a reducing thiol derivative in which said reducingthiol is a compound of formula: ##STR4## wherein R¹, R² and R³ areindependently a straight or branched lower alkyl which may besubstituted by hydroxy, lower alkyloxy or lower alkyl carbonyloxy. 9.The pharmaceutical composition according to claim 8, in which saidreducing thiol derivative is selected from the group consisting ofdiethyl dithiocarbamate, 1-methyl-1H-tetrazol-5-yl-thiol and1-(2-hydroxyethyl)-1H-tetrazol-5-yl-thiol.
 10. The pharmaceuticalcomposition according to claim 8, in which the surface of said fineparticles has a lipophilic and positively charged phase.
 11. Thepharmaceutical composition according to claim 8 in which said fineparticles include a lipophilic and positively charged substance in anamount of about 1 to 30% by mole of said fine particles.
 12. Thepharmaceutical composition according to claim 8 in which said fineparticles are liposomes.
 13. The pharmaceutical composition according toclaim 12, in which said liposomes include natural or semisyntheticphospholipid.
 14. The pharmaceutical composition according to claim 8,in which said fine particles are produced by dissolving a fine particleforming material and a lipophilic and positively charged substance in anamount of about 1 to 30% by mole of the total component in an organicmedium, and then, in case of an anti-cataract agent being lipophilic,further dissolving said anti-cataract agent in the resultant organicmedium, and in case of an anti-cataract agent being water-soluble,dissolving said anti-cataract agent in an aqueous medium and adding theresultant aqueous medium to the resultant organic medium, and in eachcase forming fine particles to obtain fine particles of which thesurface has a lipophilic and positively charged phase.
 15. Apharmaceutical composition for treatment of ophthalmic disorders whichcomprises an agent for treatment of ophthalmic disorders, included infine particles selected from the group consisting of emulsions,nanocapsules, albumin microspheres and liposomes, the surface of saidfine particles having a lipophilic and positively charged phase.
 16. Thepharmaceutical composition according to claim 15, in which said fineparticles include a lipophilic and positively charged substance in anamount of about 1 to 30% by mole of said fine particles.
 17. Thepharmaceutical composition according to claim 15, in which said fineparticles are liposomes.
 18. The pharmaceutical composition according toclaim 17, in which said liposomes include natural or semisyntheticphospholipid.
 19. The pharmaceutical composition according to claim 15in which said fine particles are produced by dissolving a fine particleforming material and a lipophilic and positively charged substance in anamount of about 1 to 30% by mole of the total component in an organicmedium, and then, in case of said agent for treatment of ophthalmicdisorders being lipophilic, further dissolving said agent in theresultant organic medium, and in case of said agent for treatment ofophthalmic disorders being water-soluble, dissolving said agent in anaqueous medium, and in each case forming fine particles to obtain fineparticles of which the surface has a lipophilic and positively chargedphase.
 20. A method for treatment of cataract which comprisesadministering to a subject in need of such treatment, a radicalscavenger in an amount effective in treatment of cataract comprising adisulfide derivative of formula: ##STR5## wherein R¹ and R² areindependently a straight or branched lower alkyl which may besubstituted by hydroxy, lower alkyloxy or lower alkyl carbonyloxy. 21.The method according to claim 20, in which said disulfide derivative isdisulfiram.
 22. A method for treatment of cataract which comprisesadministering to a subject in need of such treatment, a radicalscavenger in an amount effective in treatment of cataract comprising areducing thiol derivative in which said reducing thiol is a compound offormula: ##STR6## wherein R¹ and R² and R³ are independently a straightor branched lower alkyl which may be substituted by hydroxy, loweralkyloxy or lower alkyl carbonyloxy.
 23. The method according to claim22, in which said reducing thiol derivative is selected from the groupconsisting of diethyl dithiocarbamate, 1-methyl-1H-tetrazol-5-yl-thioland 1-(2-hydroxyethyl)-1H-tetrazol-5-yl-thiol.